Nile Red Quantifier: a novel and quantitative tool to study lipid accumulation in patient-derived circulating monocytes using confocal microscopy

• Published in: Journal of lipid research Vol. 58; no. 11; pp. 2210 - 2219
• Main Authors: Schnitzler, Johan G., Bernelot Moens, Sophie J., Tiessens, Feiko, Bakker, Guido J., Dallinga-Thie, Geesje M., Groen, Albert K., Nieuwdorp, Max, Stroes, Erik S.G., Kroon, Jeffrey
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 01.11.2017; Journal of Lipid Research; The American Society for Biochemistry and Molecular Biology; Elsevier
• Subjects: analytic method; Arteriosclerosis; bleaching; Blood Chemical Analysis - methods; cell isolation; cholesterol and trafficking; Clinical Medicine; Computer applications; Confocal microscopy; controlled study; correlation coefficient; fat droplet; fluorescence and confocal imaging; glycoprotein p 15095; human; human cell; Humans; Hypercholesterolemia; Inflammation; Klinisk medicin; lipid analysis; lipid droplets; Lipid Droplets - metabolism; Lipids; Lipids - blood; Low density lipoprotein; mathematical analysis; membrane permeability; Methods; Microscopy; Microscopy, Confocal; monocyte; Monocytes; Monocytes - metabolism; morphology; Nile Red Quantifier; Oxazines - metabolism; Phospholipids; priority journal; protein binding; protein expression; reproducibility; ultracentrifugation; vascular cell adhesion molecule 1; fluorescence and confocal imaging; lipid droplets; cholesterol and trafficking; methods
• ISSN: 0022-2275; 1539-7262
• DOI: 10.1194/jlr.D073197

The inflammatory profile of circulating monocytes is an important biomarker for atherosclerotic plaque vulnerability. Recent research revealed that peripheral lipid uptake by monocytes alters their phenotype toward an inflammatory state and this coincides with an increased lipid droplet (LD) content. Determination of lipid content of circulating monocytes is, however, not very well established. Based on Nile Red (NR) neutral LD imaging, using confocal microscopy and computational analysis, we developed NR Quantifier (NRQ), a novel quantification method to assess LD content in monocytes. Circulating monocytes were isolated from blood and used for the NR staining procedure. In monocytes stained with NR, we clearly distinguished, based on 3D imaging, phospholipids and exclusively intracellular neutral lipids. Next, we developed and validated NRQ, a semi-automated quantification program that detects alterations in lipid accumulation. NRQ was able to detect LD alterations after ex vivo exposure of isolated monocytes to freshly isolated LDL in a time- and dose-dependent fashion. Finally, we validated NRQ in patients with familial hypercholesterolemia and obese subjects in pre- and postprandial state. In conclusion, NRQ is a suitable tool to detect even small differences in neutral LD content in circulating monocytes using NR staining.